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Cell freezing liquid not containing protein and serum and preparation method thereof

A technology for cryopreservation and cells, which is applied to the field of cell cryopreservation without protein and serum and its preparation, which can solve the problems of the safety of contaminated biological products, animal injury, risks, etc., to avoid programmed cooling and reduce pollution. , the effect of large batch differences

Inactive Publication Date: 2017-08-01
SUZHOU NEW CELL & MOLECULAR BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, this method has several flaws
For example, protein and even potential pathogen contamination in serum will pose risks to the safety of biological products; in addition, fetal bovine serum will cause certain potential harm to animals, and may encounter problems that cannot pass European and American ethical audits

Method used

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  • Cell freezing liquid not containing protein and serum and preparation method thereof
  • Cell freezing liquid not containing protein and serum and preparation method thereof
  • Cell freezing liquid not containing protein and serum and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Preparation of cell cryopreservation solution without protein and serum:

[0033] (1) In a 1000ml beaker, add 600ml of double distilled water, slowly add 10g of polyvinyl alcohol, and stir to fully dissolve it.

[0034] (2) Gradually add 32 g of glucose, 1.7 g of sodium chloride, 0.06 g of sodium dihydrogen phosphate, 0.5 g of disodium hydrogen phosphate, 0.5 g of 4-hydroxyethylpiperazineethanesulfonic acid (HEPES), 0.04 g of ethylene dihydrogen phosphate Alcohol bis(2-aminoethyl ether)tetraacetic acid (EGTA), stir to fully dissolve.

[0035] (3) Add 100ml of dimethyl sulfoxide (DMSO), stir to dissolve it fully, and set the volume to 1000ml.

[0036] (4) Under aseptic conditions, the above mixed solution is first clarified and filtered with a 0.45um filter membrane, and then 0.22um filter sterilized under aseptic conditions to obtain a serum- and protein-free cell cryopreservation solution.

Embodiment 2

[0038] Preparation of cell cryopreservation solution without protein and serum:

[0039] (1) In a 1000ml beaker, add 600ml of double distilled water, slowly add 30g of polyvinyl alcohol, and stir to fully dissolve it.

[0040] (2) Gradually add 30 g of glucose, 1.7 g of sodium chloride, 0.06 g of sodium dihydrogen phosphate, 0.5 g of disodium hydrogen phosphate, 0.5 g of 4-hydroxyethylpiperazineethanesulfonic acid (HEPES), 0.04 g of ethylene dihydrogen phosphate Alcohol bis(2-aminoethyl ether)tetraacetic acid (EGTA), stir to fully dissolve.

[0041] (3) Add 100ml of dimethyl sulfoxide (DMSO), stir to dissolve it fully, and set the volume to 1000ml.

[0042](4) Under aseptic conditions, the above mixed solution is first clarified and filtered with a 0.45um filter membrane, and then 0.22um filter sterilized under aseptic conditions to obtain a serum- and protein-free cell cryopreservation solution.

Embodiment 3

[0044] Preparation of cell cryopreservation solution without protein and serum:

[0045] (1) In a 1000ml beaker, add 600ml of double distilled water, slowly add 20g of polyvinyl alcohol, and stir to fully dissolve it.

[0046] (2) Gradually add 30 g of glucose, 1.7 g of sodium chloride, 0.06 g of sodium dihydrogen phosphate, 0.5 g of disodium hydrogen phosphate, 0.5 g of 4-hydroxyethylpiperazineethanesulfonic acid (HEPES), 0.04 g of ethylene dihydrogen phosphate Alcohol bis(2-aminoethyl ether)tetraacetic acid (EGTA), stir to fully dissolve.

[0047] (3) Add 100ml of dimethyl sulfoxide (DMSO), stir to dissolve it fully, and set the volume to 1000ml.

[0048] (4) Under aseptic conditions, the above mixed solution is first clarified and filtered with a 0.45um filter membrane, and then 0.22um filter sterilized under aseptic conditions to obtain a serum- and protein-free cell cryopreservation solution.

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Abstract

The invention relates to cell freezing liquid not containing protein and serum and a preparation method thereof. The cell freezing liquid comprises polyvinyl alcohol, glucose, sodium chloride, sodium dihydrogen phosphate, disodium hydrogen phosphate, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, ethyleneglycol bis(2-aminoethylether)tetraacetic acid, dimethyl sulfoxide, and the balance of double distilled water. The cell freezing liquid has the advantages that the cell freezing liquid with single component and clear chemical components is established, the traditional serum is replaced, the possibility of pollution by viruses, bacteria, mycoplasma and the like is avoided, and the batch difference due to serum is reduced; a method of direct refrigerating in a -80 DEG C refrigerator is established, the freezing time is longer than or equal to 3 years, the procedure cooling step is not needed, and the freezing box is not used; compared with the traditional method, the operation is simple and convenient, and the safety is high; the survival rate and high activity of the frozen cells are guaranteed, the effect is better in comparison with the traditional serum, the serum is replaced, and the price is lower.

Description

[0001] Technical field: [0002] The invention belongs to the technical field of cell preservation, and in particular relates to a cell cryopreservation solution free of protein and serum and a preparation method thereof. [0003] Background technique: [0004] Cell cryopreservation is one of the main methods of cell preservation. Using cryopreservation technology to store cells in -196°C liquid nitrogen at low temperature can temporarily remove the cells from the growth state and preserve their cell characteristics, so that the cells can be revived for experiments when needed. Moreover, moderately preserving a certain amount of cells can prevent the cells from being lost due to contamination of the cells being cultured or other accidents, which plays a role in cell preservation. In addition, some cells can also be purchased, donated, exchanged and shipped in the form of cell freezing. The classic protocol for cryopreservation of cells is often applied to the cryopreservation...

Claims

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Application Information

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IPC IPC(8): A01N1/02C12N5/071
CPCA01N1/0221A01N1/0226A01N1/0284C12N5/0018C12N2500/12C12N2500/30C12N2500/34
Inventor 杨勇刘永双谢慧慧
Owner SUZHOU NEW CELL & MOLECULAR BIOTECH CO LTD
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